A plant-based medicinal food inhibits the growth of human gastric carcinoma by reversing epithelial-mesenchymal transition via the canonical Wnt/β-catenin signaling pathway

Abstract

Background: Natural products, especially those with high contents of phytochemicals, are promising alternative medicines owing to their antitumor properties and few side effects. In this study, the effects of a plant-based medicinal food (PBMF) composed of six medicinal and edible plants, namely, Coix seed, Lentinula edodes, Asparagus officinalis L., Houttuynia cordata, Dandelion, and Grifola frondosa, on gastric cancer and the underlying molecular mechanisms were investigated in vivo.

Methods: A subcutaneous xenograft model of gastric cancer was successfully established in nude mice inoculated with SGC-7901 cells. The tumor-bearing mice were separately underwent with particular diets supplemented with three doses of PBMF (43.22, 86.44, and 172.88 g/kg diet) for 30 days. Tumor volumes were recorded. Histopathological changes in and apoptosis of the xenografts were evaluated by hematoxylin and eosin staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining, respectively. Serum levels of TNF-α, MMP-2, and MMP-9 were detected by enzyme-linked immunosorbent assay. The mRNA expression levels of β-catenin, GSK-3β, E-cadherin, N-cadherin, MMP-2/9, Snail, Bax, Bcl-2, Caspase-3/9, and Cyclin D1 were evaluated via real-time quantitative polymerase chain reaction. The protein expression levels of GSK-3β, E-cadherin, N-cadherin, and Ki-67 were determined by immunohistochemistry staining.

Results: PBMF treatment efficiently suppressed neoplastic growth, induced apoptosis, and aggravated necrosis in the xenografts of SGC-7901 cells. PBMF treatment significantly decreased the serum levels of MMP-2 and MMP-9 and significantly increased that of TNF-α. Furthermore, PBMF treatment notably upregulated the mRNA expression levels of GSK-3β, E-cadherin, Bax, Caspase-3, and Caspase-9 but substantially downregulated those of β-catenin, N-cadherin, MMP-2, MMP-9, Snail, and Cyclin D1 in tumor tissues. The Bax/Bcl-2 ratio was upregulated at the mRNA level. Moreover, PBMF treatment remarkably increased the protein expression levels of GSK-3β and E-cadherin but notably reduced those of Ki-67 and N-cadherin in tumor tissues.

Conclusions: The PBMF concocted herein exerts anti-gastric cancer activities via epithelial-mesenchymal transition reversal, apoptosis induction, and proliferation inhibition. The underlying molecular mechanisms likely rely on suppressing the Wnt/β-catenin signaling pathway.

Keywords: Epithelial–mesenchymal transition; Gastric cancer; Medicinal food; Wnt/β–catenin signaling pathway.

Fig. 1

PBMF inhibited SGC-7901 xenograft tumors growth in nude mice. a Dynamic changes of tumor weights in each group during different treatments for 30 days as designed. Tumor weights were recorded once every 3 day. b Representative photographs of tumors isolated from mice in each group after sacrifice. c Dynamic changes of tumor volumes in each group during entire experiment. Tumor volumes were measured by vernier calipers and recorded once every 3 day. d All tumors were weighed on day 30. Tumor weights of 5-Fu group as well as low-, medium-dose and high-dose group are significantly less than that of model control group. Results are represented as mean weight ± SD. ^**p < 0.01 vs. model control. (Each part of the multi-panel Fig. 1 see Additional file 1)

Fig. 2

Histopathological changes of SGC-7901 xenograft tumor tissues. Tumor necrosis areas were determined by H&E staining and observed under light microscope (200×). Red arrows refer to the necrosis areas. (Each part of the multi-panel Fig. 2 see Additional file 2)

Fig. 3

Effects of PBMF on apoptosis induction of tumor cells in SGC-7901 xenograft tumor model. a TUNEL staining of xenograft tumor tissues was performed and observed under fluorescent microscope (200×). Nuclei with green fluorescence were considered as TUNEL positive cells, while nuclei with blue fluorescence were consider as total cancer cells. b Number of TUNEL positive cells was calculated at high magnification in five random fields under a fluorescent microscope. Results were represented as mean ± SD. ^*p < 0.05 vs. model control; ^**p < 0.01 vs. model control. (Each part of the multi-panel Fig. 3 see Additional file 3)

Fig. 4

Effects of PBMF on serum levels of TNF-α, MMP-2 and MMP-9 in nude mice. Results were represented as mean ± SD. ^*p < 0.05 vs. model control group; ^**p < 0.01 vs. model control. (Each part of the multi-panel Fig. 4 see Additional file 4)

Fig. 5

RT-PCR showing the effects of PBMF on mRNA expression involved in EMT and Wnt/β-catenin signaling pathway in tumor tissues. Results were represented as mean ± SD. ^**p < 0.01 vs. model control. (Each part of the multi-panel Fig. 5 see Additional file 5)

Fig. 6

RT-PCR showing the effects of PBMF on mRNA expression involved in apoptosis and proliferation in tumor tissues. Results were represented as mean ± SD. ^*p < 0.05 vs. model control group; ^**p < 0.01 vs. model control group. (Each part of the multi-panel Fig. 6 see Additional file 6)

Fig. 7

Effects of PBMF on protein expressions of GSK-3β, E-cadherin, N-cadherin and Ki-67 in tumor tissues. a Immunohistochemistry staining of GSK-3β, E-cadherin, N-cadherin and Ki-67 proteins among each group was performed and observed under light microscope (200×). GSK-3β proteins were markedly expressed in the cytoplasm and nucleus; E-cadherin and N-cadherin proteins were markedly expressed in cytomembrane and cytoplasm; Ki-67 proteins were markedly expressed in the nucleus. Red arrows refer to positive staining with pale brown. Semi-quantitative analysis of the intensity of GSK-3β-positive (b), E-cadherin-positive (c), N-cadherin-positive (d) and Ki-67-positive (e) staining was conducted using IOD/area value. ^**p < 0.01 vs. model control. (Each part of the multi-panel Fig. 7 see Additional file 7)